SIGNAL TRANSDUCTION BY PROTEIN KINASE C IN MAMMALIAN CELLS

1. The past two decades have witnessed great advances in our understanding of the role of protein kinase C (PKC) in signal transduction. The Ca²⁺-activated, phospholipid-dependent protein kinase discovered by Nishizuka's group in 1977 is now a family of at least 11 isoforms. Protein kinase C isoforms exist in different proportions in a host of mammalian cells and each isoform has a characteristic subcellular distribution in each cell type. 2. Stimulation of a specific PKC is oform often causes redistribution of the isoform from one subcellular compartment to another compartment where it complexes with and phosphorylates a specific protein substrate. 3. The interaction of a specific PKC isoform with its protein substrate may directly activate a specific function of the cell or may trigger a cascade of protein kinases that ultimately stimulates a specific response in differentiated cells or regulates growth and proliferation in undifferentiated cells.     

Emerging targets for the pharmacology of learning and memory.

Learning and memory are dynamic processes associated with modifications in morphology, biochemistry and physiology of the nervous system, that can be analysed at different levels of biological organization. Within this context, changes correlated with cognitive processes are identifiable at distinct cellular and molecular loci of the nervous system. Synaptic plasticity represents an experience-dependent alteration of neuronal properties that subserve learning and memory, and for which a neuronal network can be considered as a candidate for being part of an engram. Multiple molecular systems at various levels of the signal transduction cascade, such as those involving receptors, second messengers and gene expression, may be linked to synaptic remodelling during cognitive processes. After a brief overview of the various facets of memory and the higher levels of cerebral organization (with special attention to the hippocampus), this review is devoted to the presentation of some novel potential therapeutic targets within the signal transduction cascade that might be considered for rational pharmacological intervention to improve learning and memory in both diseased and healthy individuals.     

Divergence and complexities in DAG signaling: looking beyond PKC

For many years protein kinase C (PKC) has been the subject of extensive studies as a molecular target for the treatment of cancer and other diseases. To better define the role of PKC isozymes in the control of cell proliferation, survival and transformation, the examination of PKC-mediated signal transduction pathways by isozyme-specific intervention has become essential. However, issues related to the selectivity of activators and inhibitors of PKC isozymes, in addition to convoluted cross-talks between phorbol ester-regulated pathways, have greatly complicated our understanding of PKC-mediated responses. An additional level of complexity is provided by the fact diacylglycerol (DAG) signals can be transduced by phorbol ester receptors other than PKC. These receptors include chimaerins, RasGRPs, MUNC13s, PKD (PKC mu) and DAG kinases beta and gamma. Thus, it is conceivable that some of the effects that were originally attributed to PKC isozymes in response to phorbol esters might be mediated by PKC-independent pathways. A key issue for the design of novel therapeutic strategies that target PKC isozymes is a comprehensive analysis of isozyme-specific signal transduction pathways in different cell types and the development of pharmacological and molecular tools that can distinguish between the various PKC and 'non-PKC' phorbol ester receptors.     

Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension

Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca(2+)](i), Ca(2+)-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin-myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca(2+)](i) and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca(2+)-dependent and Ca(2+)-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca(2+) antagonist-resistant forms of hypertension.     

Critical targets of protein kinase C in differentiation of tumour cells.

The ultimate target of pharmacological research is to find new drugs for treating human diseases such as cancer. Agents causing differentiation and thus growth arrest should be particularly useful in this regard. A potential target for such anticancer therapy is the enzyme family protein kinase C (PKC), which is involved in the transduction of signals for cell proliferation, differentiation, and apoptosis. Our recent work showing the induction of differentiation in melanoma cells by an activator of one PKC isoform, PKCdelta, touches on several important areas of investigation, which will form the basis of this review: the role of individual isoforms of PKC, their downstream targets and their specific substrates, the mechanism of activation of specific genes involved in the differentiation process, and the molecular basis for the morphological changes associated with differentiation. The central role that PKC plays in these processes points to the need for a greater understanding of the signalling pathways utilized by individual isoforms of this family of enzymes.     

The aging brain, a key target for the future: The protein kinase C involvement

The brain represents the primary centre for the regulation and control of all our body activities, receiving and interpreting sensory impulses and transmitting information to the periphery. Most importantly, it is also the seat of consciousness, thought, emotion and especially memory, being in fact able to encode, store and recall any information. Memory is really what makes possible so many of our complex cognitive functions, including communication and learning, and surely without memory, life would lose all of its glamour and purpose. Age-associated mental impairment can range in severity from forgetfulness at the border with pathology to dementia, such as in Alzheimer's disease. In recent years, one of the most relevant observations of research on brain aging relates to data indicating that age-related cognitive decline is not only due to neuronal loss, as previously thought; instead, scientists now believe that age-associated functional changes have more to do with the dysfunctions occurring over time. Within this context a prominent role is certainly played by signal transduction cascades which guarantee neuronal cell to elaborate coordinated responses to the multiple signals coming from the outside and to adapt itself to the environmental changes and requests. This review will focus the attention on protein kinase C pathway, with a particular interest on its activation process, and on the role of protein-lipid and protein-protein interactions to selectively localize the cellular responses. Furthermore, information is emerging and will be discussed on the possibility of mRNA stabilization through PKC activation. This review will also approach the issue on how alterations of these molecular cascades may have implications in physiological and pathological brain aging, such as Alzheimer's disease.     

Yeast as a powerful model system for the study of apoptosis regulation by protein kinase C isoforms

Protein kinase C (PKC) is a family of serine/threonine kinases involved in the transduction of signals that control different cellular processes, such as cell death and proliferation. This family comprises at least 10 isoforms that regulate apoptosis in an isoformspecific manner. However, controversial data about the role of individual PKC isoforms in apoptosis regulation are frequently reported. The co-existence of several PKC isoforms in a same mammalian cell, the distinct expression profile of PKC isoforms in different cell types, and the different stimulus applied may explain such contradicting results. Therefore major advances in the understanding of the molecular mechanisms that regulate the function of PKC isoforms in apoptosis are still required. Yeast has proved to be a valuable research tool to investigate molecular aspects of apoptosis regulation. Additionally, the conservation in yeast of major functional and molecular properties of mammalian PKC isoforms favours the use of this simpler cell model to uncover relevant aspects of apoptosis regulation by this kinase family. In this review, we cover the current knowledge about the role of different PKC isoforms in apoptosis. Moreover, we discuss the contribution of yeast to unravel several controversial issues about apoptosis regulation by PKC isoforms. The exploitation of yeast cells expressing individual PKC isoforms towards the identification of isoform-specific PKC modulators is also discussed. The studies here summarised highlight that the yeast cell model system can provide valuable insights in the PKC research field.     

PKD at the crossroads of DAG and PKC signaling

Diacylglycerol (DAG) and its primary target protein kinase C (PKC) regulate many important cellular responses, yet the molecular mechanisms that control the specificity of DAG and PKC signaling are not fully understood. As such, targeting the PKC pathway for therapeutic purposes has been challenging. Protein kinase D (PKD), a novel DAG receptor, has been the subject of intense investigation in recent years. DAG regulates the intracellular localization of PKD and also activates PKD through PKC by phosphorylation. The PKC-PKD signaling cascade is crucial to PKD function in cells. Important discoveries have been made regarding the roles of PKD in cell growth, gene expression, survival, motility, protein trafficking and lymphocyte biology. This kinase is implicated in pathological processes such as cardiac hypertrophy, tumor cell proliferation and metastasis. Thus, PKD represents a novel therapeutic target for the DAG-PKC signaling network.     

PKCbetaII/HuR/VEGF: A new molecular cascade in retinal pericytes for the regulation of VEGF gene expression.

Vascular endothelial growth factor (VEGF)-induced new vessels formation is a key event in diabetic retinopathy, a severe progressive multistage pathology. Literature data indicate that protein kinase C (PKC) is involved in the control of VEGF expression, but, so far, no data are available on the molecular pathway underlying this process. Within this context, we suggest the existence of a new molecular cascade, operating in retinal bovine pericytes and involving PKCbetaII, the mRNA-stabilizing protein HuR, and VEGF. In particular we show that PKCbetaII activation is responsible, through the RNA-binding protein HuR, for the increase of VEGF protein content and its release in the medium. The specificity of the PKCbetaII involvement is confirmed by experiments performed with the LY379196 compound, a selective PKCbetaII inhibitor. Following acute high-glucose insult this pathway seems still functioning, suggesting that a brief exposure to glucose does not compromise this molecular cascade in pericytes. A better understanding on this new pathway could open novel opportunities for the development of innovative pharmacological therapies useful in pathologies where VEGF plays a key role such as in diabetic retinopathy.     

Therapeutic potential of natural compounds that regulate the activity of protein kinase C

Protein kinase C (PKC) is a family of serine/threonine kinases that regulates a variety of cell functions including proliferation, gene expression, cell cycle, differentiation, cytoskeletal organization, cell migration, and apoptosis. The PKC signal transduction cascade coordinates complex physiological events including normal tissue function and repair. Disruption of the cellular environment through genetic mutation, disease, injury, or exposure to pro-oxidants, alcohol, or other insults can induce pathological PKC activation. Aberrant PKC activation can lead to diseases of cellular dysregulation such as cancer and diabetes. Can aberrant activation of PKC be reversed? Even 25 years after the identification of PKC, therapeutic regulation of PKC activity remains an emerging field. Because the function of each isoform remains to be elucidated, isoform specific control of gene expression is a current challenge. Natural compounds are important regulators of PKC activity, with both preventive and therapeutic efficacy. Antioxidants including vitamin A (retinoids), vitamin C (ascorbic acid) and vitamin E (tocopherols) show promise for reversal of PKC activation. beta-carotene and retinoids function as anticarcinogenic agents and antagonize the biological effects of pro-oxidants on PKC. Vitamin E reverses the deleterious effects of hyperglycemia and diabetes by down-regulating PKC activity. Antioxidants in red wine provide cardioprotective effects. However, alcohol consumption also induces oxidative stress and disrupts PKC and retinoid function in the fetus and the adult. This review examines modulation of PKC activity by natural compounds and pharmacologic analogues which can be used effectively to prevent or treat common diseases associated with aberrant activation of PKC.     

Gi-mediated activation of the p42/p44 mitogen-activated protein kinases by receptor mimetic basic secretagogues is abrogated by inhibitors of endocytosis

Exocytosis in mast cells, effector cells of allergic and inflammatory reactions, can be activated, in a receptor-independent manner, by a family of polycationic molecules (e.g. the Basic Secretagogues of mast cells) that activate directly heterotrimeric G-proteins that control exocytosis. We have recently shown that pertussis toxin (Ptx)-sensitive Gi-protein(s), activated directly by Basic Secretagogues, also stimulate protein tyrosine phosphorylation and activation of the p42/p44 MAP kinases, via a mechanism that involves protein kinase C (PKC), phosphatidylinositol-3-kinase and Ca2+ as intermediates (J. Pharmacol. Exp. Ther. 289 (1999) 1654). In this paper, we have investigated the role of endocytosis in this receptor-independent, G-protein-mediated signaling. Using mechanistically distinct inhibitors of clathrin-mediated endocytosis, we demonstrate that protein tyrosine phosphorylation and activation of p42/p44 MAP kinases are endocytosis-dependent. In contrast, Gi-stimulated exocytosis is unaffected. We show further that Gi activation results in recruitment of clathrin from the cytosol to the plasma membrane. Taken together, our results indicate that signal transduction between G-proteins and the components of the MAP kinase activation cascade is dependent on clathrin-mediated endocytosis and can occur independently of a 7 TM cell surface receptor.     

Potential role of PKC inhibitors in the treatment of hematological malignancies

The serine/threonine protein kinase C (PKC) family, the main target of tumor-promoting phorbol esters, is functionally associated to cell cycle regulation, cell survival, malignant transformation, and tumor angiogenesis. Although PKC isozymes represent an attractive target for novel anticancer therapies, our knowledge of PKC in tumorigenesis is still only partial and each PKC isoform may contribute to tumorigenesis in a distinct way. Specifically, PKC isoforms have wide and different roles, which vary depending on expression levels and tissue distribution, cell type, intracellular localization, protein-protein and lipid-protein interactions. Although PKC activation has been linked to tumor cell growth, motility, invasion and metastasis, other reports have shown that some PKC isoforms can also have opposite effects. Therefore, it will be necessary to analyze the relative contribution of each PKC isozymes in the development and progression of different tumors in order to identify therapeutic opportunities, using either PKC inhibitors or PKC activators as molecular tools of investigation. This minireview is focussed on the role of PKC signaling and on the perspective of PKC inhibition in hematological malignancies.     

Curcumin-induced degradation of PKCδ is associated with enhanced dentate NCAM PSA expression and spatial learning in adult and aged Wistar rats

Polysialylation of the neural cell adhesion molecule (NCAM PSA) is necessary for the consolidation processes of hippocampus-based learning. Previously, we have found inhibition of protein kinase C delta (PKCδ) to be associated with increased polysialyltransferase (PST) activity, suggesting inhibitors of this kinase might ameliorate cognitive deficits. Using a rottlerin template, a drug previously considered an inhibitor of PKCδ, we searched the Compounds Available for Purchase (CAP) database with the Accelrys^® Catalyst programme for structurally similar molecules and, using the available crystal structure of the phorbol-binding domain of PKCδ, found that diferuloylmethane (curcumin) docked effectively into the phorbol site. Curcumin increased NCAM PSA expression in cultured neuro-2A neuroblastoma cells and this was inversely related to PKCδ protein expression. Curcumin did not directly inhibit PKCδ activity but formed a tight complex with the enzyme. With increasing doses of curcumin, the Tyr¹³¹ residue of PKCδ, which is known to direct its degradation, became progressively phosphorylated and this was associated with numerous Tyr¹³¹-phospho-PKCδ fragments. Chronic administration of curcumin in vivo also increased the frequency of polysialylated cells in the dentate infragranular zone and significantly improved the acquisition and consolidation of a water maze spatial learning paradigm in both adult and aged cohorts of Wistar rats. These results further confirm the role of PKCδ in regulating PST and NCAM PSA expression and provide evidence that drug modulation of this system enhances the process of memory consolidation.     

Protein Kinase C and its Inhibitors in the Regulation of Inflammation: Inducible Nitric Oxide Synthase as an Example

Protein kinase C (PKC) is a family of ten isoenzymes that play a crucial role in cellular signal transduction. Studies with PKC knockout animals have revealed that many of the isoenzymes are involved in cell growth, proliferation and differentiation. Several PKC isoenzymes have also been shown to be important mediators in inflammation and immunity, particularly in lymphocyte responses. However, less is known about the role of PKC in the regulation of the expression of inflammatory genes. In inflammatory processes, nitric oxide is primarily produced by inducible nitric oxide synthase (iNOS) in inflammatory cells, such as macrophages. In innate immunity, nitric oxide functions as an effector molecule towards the infectious organisms. Increased levels of nitric oxide are also produced by inflammatory and tissue cells in inflammatory diseases, such as asthma and arthritis. In this MiniReview, the role of PKC isoenzymes in the pathogenesis and as a potential drug target in inflammation will be discussed presenting iNOS as an example of an inflammatory gene regulated by the pleiotropic PKC signalling pathway.     

Vitamin D deficiency-induced hypertension is associated with vascular oxidative stress and altered heart gene expression

S-nitrosylation is a ubiquitous protein modification in redox-based signaling and forms S-nitrosothiol from nitric oxide (NO) on cysteine residues. Dysregulation of (S)NO signaling (nitrosative stress) leads to impairment of cellular function. Protein kinase C (PKC) is an important signaling protein that plays a role in the regulation of vascular function, and it is not known whether (S)NO affects PKC's role in vascular reactivity. We hypothesized that S-nitrosylation of PKC in vascular smooth muscle would inhibit its contractile activity. Aortic rings from male C57BL/6 mice were treated with auranofin or 1-chloro-2,4-dinitrobenzene (DNCB) as pharmacological tools, which lead to stabilize S-nitrosylation, and propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO) as NO donors. Contractile responses of aorta to phorbol-12,13-dibutyrate, a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. S-nitrosylation of PKCα was increased by auranofin or DNCB and CysNO as compared with control protein. Augmented S-nitrosylation inhibited PKCα activity and subsequently downstream signal transduction. These data suggest that PKC is inactivated by S-nitrosylation, and this modification inhibits PKC-dependent contractile responses. Because S-nitrosylation of PKC inhibits phosphorylation and activation of target proteins related to contraction, this posttranslational modification may be a key player in conditions of decreased vascular reactivity.     

Compartmentalised MAPK pathways

The mitogen-activated protein kinase (MAPK) pathway provides cells with the means to interpret external signal cues or conditions, and respond accordingly. This cascade regulates many cell functions such as differentiation, proliferation and migration. Through modulation of both the amplitude and duration of MAPK signalling, cells can control their responses to the multiple activators of the pathway. In addition, recent work has highlighted the importance of the cellular compartment from which the signalling occurs. Cells have developed intricate systems that enable them to localise MAPK components to specific subcellular domains in response to a particular stimulus. Consequently, different factors can activate the same kinase in separate locations. Crucial to this ability are molecular scaffolds, which act as signalling modules for MAPKs, confining them to the desired compartment. The participation of the MAPK network in fundamental physiological processes, such as cell proliferation and inflammation, and the derangement of the homeostasis that occurs in disease processes, renders MAPK a highly desirable target for therapeutic intervention. As we enhance our comprehension of scaffolds and other regulatory molecules, novel targets for drug design may be discovered that will afford selective and specific MAPK modulation.     

Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development.

Protein kinase C (PKC) is a family of enzymes that play a ubiquitous role in intracellular signal transduction. Our understanding of the precise role of PKC has evolved considerably as a result of improved methodology and a better understanding of the signal transduction pathways. A number of primary pathways previously attributed to PKC have been re-examined and found to involve other kinases as our understanding of the PKC isozymes has evolved. PKC isozymes appear to play distinct, and in some cases opposing roles in the transduction of intracellular signals. The development of potent and selective PKC inhibitors, including isozyme-selective inhibitors, has opened new avenues for biochemical and pharmaceutical studies. The role of PKC in some of the pathways relevant to cardiovascular, peripheral microvascular, CNS, oncology, immune and infectious disease states are surveyed. A survey of the current generation of potent and selective ATP-competitive inhibitors is provided. The progress of PKC inhibitors currently in clinical development, including LY333531, ISIS 3521 (CGP 64128A), bryostatin 1, GF109203x, Ro 32-0432 and Ro 31-8220, Go 6976 and Go 7611, CPR 1006, and balanol (SPC 100840) are discussed.     

mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C

Potentiation of ionotropic glutamate receptor activity by metabotropic glutamate receptors (mGluRs) is thought to modulate activity at glutamatergic synapses in the hippocampus. However, the precise pathway by which this modulation occurs is not well understood. The present study tests the hypothesis that mGluR1-mediated potentiation of N-methyl-D-aspartate receptors (NMDARs) occurs via a phospholipase C (PLC)-initiated cascade. NMDAR functional activity was examined by whole-cell recording from Xenopus oocytes expressing recombinant NMDARs and mGluR1alpha. The mGluR1 agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) significantly potentiated NMDA-elicited currents. mGluR1alpha-mediated potentiation of NMDA responses was eliminated by the PLC inhibitor U-73122. Buffering of intracellular Ca2+ by BAPTA-AM or depletion of intracellular Ca2+ by the Ca2+/ATPase inhibitor thapsigargin greatly reduced ACPD potentiation. ACPD potentiation was reduced by the specific protein kinase C (PKC) inhibitor Ro-32-0432 and eliminated by the broad spectrum kinase inhibitor staurosporine. ACPD produced no further potentiation after potentiation of NMDARs by the PKC-activating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Thus, Group I mGluRs potentiate NMDA responses via activation of PLC; at least part of the potentiation is due to rise in intracellular Ca2+ and stimulation of PKC. Cytochalasin D, which disrupts the actin cytoskeleton, blocked ACPD-elicited chloride currents and ACPD-induced potentiation of NMDAR currents, consistent with a role for cytoskeletal protein(s) in the signaling pathway. As Group I mGluRs are localized to the perisynaptic region in juxtaposition to NMDARs at glutamatergic synapses, mGluR-mediated potentiation of NMDAR activity may play a role in synaptic transmission and plasticity including LTP.